Regulated trophoblast migration is indispensable for normal placentation. Shallow trophoblast invasion results in pregnancy complications, including preeclampsia, intrauterine growth restriction, and preterm delivery. Invasive trophoblasts, called spiral artery-trophoblast giant cells (SpA-TGCs), maintain connectivity and exhibit an epithelial to endothelial-like transformation when replacing maternal endothelial cells. The mechanism that directs collective and directional cell movement in SpA-TGCs remains unknown. The proposed project aims to study how planar cell polarity (PCP) signaling in cooperation with cannabinoid signaling regulates organized trophoblast migration for placentation. Studies are aimed to reveal the impact of disrupted protein complex of cannabinoid receptor 1 (CB1) and VANGL2, a core component of PCP, signaling in placentation, and downstream signaling pathways regulating trophoblast migration. In addition, the results will show the advantage of collective SpA-TGC migration and identify signaling pathways critical for migration. Understanding the molecular basis of SpA-TGC migration may provide a new strategy to treat preeclampsia. We propose that PCP and CB1 signaling suppresses random cell protrusions to direct collective trophoblast migration in placentation. Our specific aims are to test the hypotheses: (1) that PCP signaling is critical for SpA-TGC migration, and (2) that CB1 and PCP signaling converges to regulate organized migration of TS cell movement via Rac1. Our preliminary results show that the deletion of Vangl2 or Cnr1 (encoding CB1) compromises trophoblast stem (TS) cell migration in vitro. To study the role of VANGL2 and CB1 in trophoblast invasion in vivo, we propose to create mouse models with conditional deletion of Vangl2 and Cnr1 in SpA-TGCs using a Tpbpa-Cre driver (Vangl2f/fTpbpacre/+ mice), and examine trophoblast differentiation, migration, maternal endothelial cells replacement and pregnancy outcomes in these mice. We expect that SpA-TGCs missing Vangl2 or Cnr1 have compromised trophoblast migration and placentation. Using Cnr1 or Vangl2 deleted TS cells and mouse models, we will test if CB1 modulates PCP signaling via functional association with VANGL2. We will test the effects of CB1 agonists and antagonists on TS cell migration in WT, Cnr1-/- and Vangl2-/- TS cells in vitro, and WT and Vangl2f/fTpbpacre/+ mice in vivo. These studies will be followed by how CB1/VANGL2 protein complex suppresses random cell protrusion by inhibiting Rac1 GTPase to order directional cell migration. The results of the proposed research will unravel novel regulatory pathways in the directional migration of SpA-TGCs during placentation. The formation of a CB1/VANGL2 protein complex will uncover a new molecular function of cannabinoid and PCP signaling in placentation. The results will also be of great interest to investigators in neuroscience, as these signaling pathways are active in the brain functions. The findings may help support public policy initiatives for the nation's overall health from cannabis consumption.